Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in the release of various RNA viruses, including the primate lentiviruses. It is unclear how these components, which are normally hosted by the endo-lysosomal system, are recruited to the plasma membrane in cell types such as activated lymphocytes, where HIV-1 exits primarily by budding from the cell surface. Here we propose to study the feasibility of using evanescent field fluorescence microscopy (EF-FM), also called total internal reflection fluorescence microscopy (TIR-FM), to investigate in real time, how the plasma membrane microcompartments from which HIV-1 buds are formed. EF-FM/TIR-FM allows selective illumination of the plasma membrane and the underlying approximately 100 nm of cytoplasm. Fluorescent protein-tagged viral and cellular proteins will be utilized to determine if the plasma membrane microdomains, which serve as budding platforms for HIV-1 are assembled simultaneously with the arrival of the viral structural proteins or if they are pre-existing loci. We anticipate that EF-FM/TIR-FM will become an important tool to elucidate, with high spatial and temporal resolution, the sequence of events culminating in the release of newly formed viral particles.